Cytokinins are important plant hormones that are defined by their ability to promote cell division in plant tissue culture in the presence of auxins (Skoog et al., Science 148, 532-533, 1965). Cytokinins so far found in plants are adenine derivatives substituted at the N6-position with either an isoprenoid or an aromatic sidechain. Isoprenoid trans-zeatin (tZ) is the most abundant cytokinin. The abundance of other isoprenoid cytokinins—N6-isopentenyladenine, iP, cis-zeatin, cZ and its derivative with a saturated sidechain dihydrozeatin (DHZ), varies between plant species. While isoprenoid cytokinins are ubiquitous in plants, aromatic cytokinins, represented by N6-benzyladenine, BA, and its hydroxylated derivatives, the topolins) have only been identified, as yet, in a limited group of plant taxa (Horgan et al., Phytochemistry 14, 1005-1008, 1975; Strnad, Physiol. Plant. 101, 674-688, 1997; Strnad et al., Plant Physiol. 99, 74-80, 1992). The most abundant appears to be ortho-topolin riboside, which is present in micro-molar concentrations in poplar leaves after daybreak (Hewett et al., Planta 114, 119-129, 1973). Both families of cytokinins occur in several forms: free bases, ribosides, riboside-5′-monophosphates, 3-, 7-, 9- and O-glucosides, and amino acid conjugates.
Knowledge that cytokinins play key roles in the regulation of plant growth and differentiation led to the postulation that they have a potential utility for treating human diseases that involve dysfunctional cell proliferation and/or differentiation. The ability of cytokinin bases to induce or promote the differentiation of human cells has been demonstrated in keratinocytes (Berge et al., Ann. N. Y. Acad. Sci. 1067, 332-336, 2006) and several leukaemia cell lines, including HL-60 and K-562 (Ishii et al., Biochim. Biophys. Acta. 1643, 11-24, 2003). However, while free bases induce differentiation at relatively high concentrations (25-100 μM), their ribosides cause rapid apoptosis of leukemia cell lines in micromolar concentrations (Mlejnek, J. Cell Biochem. 83, 678-689, 2001). Cell death in HL-60 is preceded by depletion of adenosine triphosphate, activation of caspases and mitochondrial depolarization (Mlejnek, J. Cell Biochem. 83, 678-689, 2001; Ishii et al., Biochim. Biophys. Acta. 1643, 11-24, 2002). Intracellular conversion of ribosides into their monophosphates is necessary for their functioning (Meljnek and Dole{hacek over (z)}el, Toxicol. In Vitro 19, 985-990, 2005). The caspase inhibition shifts the activity of N6-isopentenyladenosine (iPR) in HL-60 to growth inhibitory and differentiating activity (Ishii et al., Biochim. Biophys. Acta. 1643, 11-24, 2002). It was recently shown that kinetin riboside (KR) could be a suitable drug candidate against multiple myeloma (Tiedemann et al., J. Clin. Invest. 118, 1750-1764, 2008). In several multiple myeloma models, KR induced rapid suppression of cyclin D1 and D2 transcription followed by cell-cycle arrest and tumour-specific apoptosis (Tiedemann et al., J. Clin. Invest. 118, 1750-1764, 2008). Cytotoxic effects of iPR, KR and N6-benzyladenosine (BAR) against human cell lines derived from solid tumours were reported by Cabelo et al. (Int. J. Cancer 120, 2744-2748, 2008), Choi et al. (Cancer Lett. 261, 37-45, 2008), Laezza et al. (Int. J. Cancer. 124, 1322-1329, 2009), Meisel et al. (FEBS Lett. 433, 265-268, 1998) and Spinola et al. (Int. J. Cancer 120, 2744-2748, 2007). Depending on the cell line and cytokinin used, the treatment resulted in cell cycle block (either G1 or G2/M phase) and/or apoptosis. The in vivo anticancer activity of iPR, KR and BAR was also demonstrated in several animal and xenograft models of cancer (Choi et al., Cancer Lett. 261, 37-45, 2008; Laezza et al., FASEB J. 20, 412-418, 2006; Tiedemann et al., J. Clin. Invest. 118, 1750-1764, 2008). iPR and BAR also showed a promising activity against diverse malignancies in a small clinical trial (Mittelman et al., Ann. N. Y. Acad. Sci. 255, 225-234, 1975).
Micromolar concentrations of cytokinin ribosides and cytokinin bases are also able to induce cell death with features of apoptosis (activation of caspase-like proteases and fragmentation of DNA) in plant cell cultures (Mlejnek and Prochazka, Planta 215, 158-166, 2002). The cell death is also preceded by depletion of adenosine triphosphate and reactive oxygen species production. In contract to their hormonal activity which requires their interaction with a membrane receptor, the intracellular conversion of cytokinins into their monophosphates is necessary for their cytotoxic effect. The cytotoxic concentrations are higher than endogenous cytokinin levels in plant tissue but yet in the concentration range used in plant biotests (Carimi et al., Planta 216, 413-421, 2003; Mlejnek et al., Plant. Cell. Environ. 26, 1723-1735, 2003; Plant. Sci. 168, 389-395, 2005). Cytotoxic activity of naturally occuring cytokinins and their analogues (Dole{hacek over (z)}al et al., Bioorg. Med. Chem. 14, 875-884, 2006; Bioorg. Med. Chem. 15, 3737-3747, 2007; CZ 294538) in mammalian and human experimental systems was demonstrated repeatedly.
It is an object of this invention to provide new anticancer and proapoptotic heterocyclic compounds derived from N6-benzyladenosine-5′-monophosphate substituted on the phenyl moiety, having improved selectivity and therapeutic effectivity index, i.e., compounds which are less toxic but highly active. Hence they can be used for development of new generation of anticancer, antimitotic and proapoptotic drugs, as well as drugs with other medical activities.